Chronic alcohol (ethanol) abuse, especially binge-type alcoholism, causes brain damage by unresolved mechanisms. Adult rats exposed to high blood alcohol levels via repeated binges incur hippocampal (HC) and entorhinal cortical (EC) neurodegeneration that is conceivably downstream of brain edema, neuroinflammation and oxidative stress (OxS). Our collaborative studies with Dr. Hee-Yong Kim at NIAAA and others, which have utilized an adult male rat model and a parallel in vitro model (rat organotypic HC-EC slice cultures), provide evidence that binge alcohol activates OxS and edema-related, phospholipid-linked pro-oxidative pathways via previously unacknowledged neuroinflammatory routes. The specific proteins activated, quite likely in different cells, include aquaporin-4 (AQP4, poly (ADP-ribose) polymerase-1 (PARP-1), and certain phospholipase A2 (PLA2) isoforms that mobilize excessive ?6 arachidonic acid (ARA) to generate OxS. Furthermore, ?3 docosahexaenoic acid (DHA), possibly via several mechanisms, is potently anti-inflammatory and neuroprotective in the cultures. In continued collaboration with Dr. Kim's laboratory, we will explore the signaling between these factors with an expanded neuroinflammatory hypothesis: PARP-1 and AQP4 are the critical 1st response proteins that govern distinct binge alcohol-dependent neuroinflammatory pathways-with PARP-1 sensing initial DNA oxidative damage/strand breaks from alcohol-generated OxS, becoming hyper-elevated, and triggering PLA2-derived excessive ARA release, while the increased AQP4 promotes brain edema, DHA loss and Akt reductions to elicit more OxS, with both paths coalescing in neurodamage; supplemented DHA then promotes antioxidative suppression of these pathways and neuroprotection via mechanisms that may involve both its free and lipid-bound forms. Three aims are proposed: the first two aims use binge alcohol-treated adult-age HC-EC slice cultures (newly developed in our current studies), while aim 3 extrapolates in vitro results to a fitting in vivo model, repetitive binge intoxication of adult male rats (Majchrowicz model)-a model in which neurodegeneration was first characterized in our laboratories and is being used by NIAAA investigators. AIM 1 examines how PARP-1, AQP4 and PLA2 are regulated by alcohol and relate to each other and to neurodegeneration, using specific inhibitors and RNA interference; AIM 2 probes supplemental DHA's mechanisms that preclude alcohol- dependent neuroinflammatory phospholipid mechanisms while achieving neuroprotection, including its membrane phospholipid incorporation as well as its metabolism to protective resolvins and neuroprotectin-1; and AIM 3 extends results from Aim 1 and 2 experiments to the rat binge model, examining our binge alcohol/PARP-1 cascade hypothesis and DHA's neuroprotective mechanism in vivo. The results could shed insight into novel neuroinflammatory/neurotoxic alcohol mechanisms, and clarify how DHA supplementation might be cognitively beneficial as adjunct therapy in alcoholism treatment.